Abstract
The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) belongs to carbohydrate binding module (CBM) family 21 with high binding affinities toward raw starches, and contains two ligand binding sites at opposite position (Site I: Trp47, Tyr83 and Tyr94; Site II: Tyr32, Phe58). Three dimensional structure reveals that two RoSBD molecules hold the same sugar ligand by different binding sites. Natural CBMs in tandem repeat show higher ligand-binding affinity than single CBM does, yet whether two cooperative RoSBDs enhance ligand binding properties remains to be investigated. In this study, dimeric RoSBD was constructed with a short peptide linker between two RoSBD units. Isothermal titration calorimetry (ITC) data indicated that dimeric RoSBD processed higher affinities toward a series of glycans than monomeric RoSBD. Moreover, site-directed mutagenesis of major ligand binding residues Y32 and W47 on each domain of dimeric RoSBD revealed determining factors by ITC analysis. In addition, in silico structure modeling was carried out by quantitative measurement of binding affinity between wild-type/mutant dimeric RoSBD and soluble starch by ITC and published X-ray crystallography data. For short glycans such as maltoheptaose (Glc7), or cyclic ligand β-cyclodextrin (βCD), two RoSBD molecules might hold on the same sugar ligand by different binding sites based on X-ray crystallography data to predict. However, according to ITC data, Y32 on the N-terminal RoSBD played a crucial role in Glc7 binding and Y32 on the C-terminal RoSBD was important for βCD binding. For long chain glycans, Y32 in both two RoSBD molecules played a crucial role in ligand binding, indicated that two binding site II of dimeric RoSBD bound to a same glycan to form RoSBD/RoSBD interface. In conclusion, RoSBD might bind to various glycans in different interaction models. The interaction model between RoSBD and glycans will greatly increase our understanding in the molecular mechanisms of CBM functions.